Genetic Analysis of virally-infected Nicotiana benthamiana
Overview of Nicotiana benthamiana Nicotiana benthamiana belongs to the genus of plants colloquially known as tobacco plants. N. benthamiana is native to northern Australia, where it grows in hilly and mountainous regions; it is a relatively short plant that springs many frail leaves and white flowers (1). This specific Nicotiana species is considered the most widely utilized model organism in experimental plant virology trials. This is because of N. benthamiana’s biological susceptibility to viral infections (2). A naturally occurring mutation in an RNA-dependent RNA polymerase gene allows viruses to easily take over the plant’s transcription machinery (3). N. benthamiana'' is becoming a more commonly utilized organism for protein production platforms and genetic sequencing techniques. Plant virus-based transient expression systems are large-scale protein production systems, yielding quickly produced and large amounts of growth factors and antibodies; virally-induced gene silencing experiments reveal the functions of various genes among many different species of plants (3). Other than information gathered from these experiements, little is known about genetic variation among ''N. benthamiana strains (2). A draft of the entire N. benthamiana genome was released recently in 2012 (3). Genetic Analysis of the N. benthamiana Genome This particular genetic study of N. benthamiana assessed the differences in transcription and gene expression between a normal plant and a virally infected plant. Researchers were interested in detecting differences in gene expression and stability between the two models by observing 16 major housekeeping genes (GAPDH, 18S, EF1α, ''SAMD, L23, UK, PP2A, APR, UB13, SAND, ACT, TUB, GBP, F-BOX, PPR, TIP41) (3). By using quantitative real-time PCR techniques, a highly accuracy, reproducible technique, one can observe the slight changes in gene expression of these housekeeping gene under different conditions. First, researchers determined references levels of gene expression for these 16 genes in a healthy N. benthamiana ''specimen. ''Several ''N. benthamiana specimens were then infected with one of these five types of''RNA viruses: tobacco necrosis virus A, beet black scorch virus, beet necrotic yellow vein virus, barley stripe mosaic virus, or potato virus X (3). Leaves from the five virally infected hosts were harvested and had their genetic content purified. After taking this genetic content and performing qPCR to amplify the genes, researchers put this genetic information through several algorithm-based statistical analyses programs: ''geNorm, NormFinder, ''and ''BestKeeper (3). The data retrieved from these programs reveals the differences in gene expression and stability between different N. benthamiana'' models. Overall, the three analyses programs indicated that the genes least affected by viral infection, therefore exhibiting high expression stability, were the L23, PP2A, ''and ''F-box genes; the genes most affected by viral infection, exhibiting the lowest expression stability, were the TUB, ACT, and'' GAPDH'' genes (3). AGO2 and RdR6, two other N. benthamiana housekeeping genes that regular RNA-based antiviral immunity, were observed in detail as well. The expression and stability of these two genes were characterized differently than the previous 16 housekeeping genes. Researchers observed that under virally infected conditions, expression AGO2 increased significantly, while expression of RdR6 barely changed at all (3). References 1) Nicotiana benthamiana. Wikipedia. http://en.wikipedia.org/wiki/Nicotiana_benthamiana. Updated August 13, 2014. Accessed October 26, 2014. 2) Goodin MM, Zaitlin D, Naidu RA, et. al. Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions. Mol Plant Microbe Interact. August 2008; 21(8): 1015-1026. doi: 10.1094/MPMI-21-8-1015. 3) Liu D, Shi L, Zhang Y, et. al. Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR. ''PLoS One. September 28, 2012; 7(9): e46451. doi: 10.1371/journal.pone.0046451